Current Topic
pubs.acs.org/biochemistry
Redox Regulation of Epidermal Growth Factor Receptor Signaling
through Cysteine Oxidation
Thu H. Truong† and Kate S. Carroll*,‡
†
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33456, United States
‡
ABSTRACT: Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine
kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation.
Moreover, gene amplification and EGFR mutations have been identified in a number of human
malignancies, making this receptor an important target for the development of anticancer drugs. In
addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR
stimulation results in the localized generation of H2O2 by NADPH-dependent oxidases. In turn,
H2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through
the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797
in the EGFR active site. In this review, we highlight recent advances in our understanding of the
mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form
the basis for the development of new therapeutic strategies for targeting this and other H2O2modulated pathways.
A
II17 stimulate H2O2 generation. Although chronic exposure to
high concentrations of H2O2 can lead to a cellular condition
known as oxidative stress, cells can also utilize H2O2 as a
secondary messenger to regulate physiological signal transduction.18−21 H2O2 modulates signaling pathways largely
through the modification of specific cysteine residues located
within redox-sensitive protein targets (Figure 2A). The direct
product of the reaction between H2O2 and a protein thiolate
(RS−) is sulfenic acid (RSOH). Such protein “sulfenylation”
can be reversed by thiol-disulfide oxidoreductases of the
thioredoxin (Trx) superfamily throughout the intracellular
milieu and, thus, constitutes a facile switch for modulating
protein function, akin to phosphorylation. Associated regulatory redox modifications of cysteine include sulfenamides,
nitrosothiols, disulfides, sulfinic acids, and sulfonic acids (Figure
2B). Over the past several years, an increasing number of
studies have demonstrated important functional roles for
protein sulfenic acids in cell signaling.22−26 In particular,
kinases and phosphatases are both known to undergo cysteinebased redox regulation,27,28 and the collective efforts of many
researchers have established that these enzymes are regulated
by endogenous H2O2 produced during EGF-mediated cell
signaling.29
In this review, we present a historical overview of EGFR and
ligand-mediated production of H2O2 as well as the molecular
mechanisms involved in redox regulation of this signaling
pathway (Figure 1). We begin by highlighting early studies
linking EGFR activation to EGF-induced H2O2 production.
Subsequently, we address the effects of redox modulation on
ctivation of receptor tyrosine kinases (RTKs) by their
respective extracellular ligands (e.g., growth factors)
initiates signaling cascades that regulate cellular proliferation,
differentiation, migration, and survival.1 Among the 58 RTKs
identified in the human genome, epidermal growth factor
(EGF) receptor (EGFR) has served as the quintessential model
for understanding RTK biology in physiological signaling and
cancer. EGFR, also known as HER1 (or erbB1), is a
transmembrane protein grouped into a subfamily that consists
of three additional, closely related receptors: HER2 (erbB2),
HER3 (erbB3), and HER4 (erbB4). EGFR is comprised of a
glycosylated extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain containing its
tyrosine kinase core. In response to ligand (e.g., EGF) binding,
EGFR forms homo- or heterodimers with other HER family
members, followed by autophosphorylation of key tyrosine
residues located within the tyrosine kinase domain.2 Once
activated, EGFR relays the signal through a variety of
downstream intracellular signaling cascades, including the
Ras/mitogen-activated protein kinase (MAPK) pathway or
the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway. Since
its discovery,3−6 EGFR has been widely studied with regard to
its physiologic and pathological settings. In particular, EGFR
and related family members have been found to be mutated or
amplified in a number of human lung and breast cancers,
making them attractive targets for the development of
therapeutics.7−9
Beginning in the 1990s, data from several research groups
demonstrated that binding of EGF to EGFR triggered the
production of endogenous hydrogen peroxide (H2O2) in cells
(Figure 1).10,11 Moreover, it was also established that other
growth factors (FGF,12 PDGF,13 VEGF,14 and insulin15),
cytokines (TNF-α12,16 and interleukin-116), and angiotensin
© 2012 American Chemical Society
Received: October 22, 2012
Revised: November 26, 2012
Published: November 27, 2012
9954
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
Figure 1. Timeline outlining key events and discoveries relating to redox regulation of EGFR signaling through cysteine oxidation.
DNA synthesis, and chemotaxis.13 These results suggested that
H2O2 might act as a signaling molecule generated in response
to growth factor stimulation. Soon thereafter, Rhee and
colleagues reported that addition of EGF to EGFR-overexpressing human epidermoid carcinoma A431 cells significantly elevated levels of intracellular reactive oxygen species
(ROS)10 as measured by 2′,7′-dichlorofluorescein diacetate
(DCFH-DA).32 Enzymes such as catalase, peroxiredoxins
(Prxs), and glutathione peroxidases (Gpxs) scavenge endogenous H2O2 by catalyzing the dismutation (catalase) or
reduction (Prxs, Gpxs) of H2O2.33,34 Introduction of exogenous
catalase in EGF-stimulated A431 cells by electroporation
attenuated the intracellular buildup of ROS, suggesting that
H2O2 was the major ROS involved in EGF-dependent signal
transduction.10 EGF-induced increases in levels of Tyr
phosphorylation of PLC-γ1, a well-characterized physiological
substrate of EGFR, were also blunted by catalase incorporation.
Although the precise role and/or target(s) of H2O2 generated
for EGFR signaling was not directly addressed in this study, the
authors proposed that inhibition of cysteine-dependent PTPs
by H2O2 may be required to increase the steady-state level of
protein Tyr phosphorylation. This landmark contribution by
Rhee and co-workers set the stage for delineating the molecular
details underlying redox regulation of EGFR signaling.
In a separate study, Goldkorn et al. reported that exogenous
H2O2 stimulated EGFR tyrosine kinase activity and increased
receptor half-life.35 Experiments performed in A549 human
lung adenocarcinoma epithelial cells and isolated membrane
fractions showed an increase in EGFR Tyr autophosphorylation
levels when treated with H2O2 (0−200 μM) and also markedly
enhanced receptor activation in conjunction with the native
EGF ligand. Pulse−chase experiments with [35S]methionine
revealed that the EGFR half-life was ∼8 h when treated with
EGF, whereas H2O2 treatment extended the receptor half-life to
18 h (combined treatment with EGF and H2O2 yielded a halflife of 12 h). Additionally, two-dimensional phosphoamino acid
analysis corroborated earlier findings30 that the phosphorylation site distribution was shifted predominantly toward Tyr
after exposure to H2O2. On the basis of these data, the authors
of this study postulated that EGF- and H2O2-induced receptor
two major pathways downstream of EGFR, Ras/MAPK and
PI3K/Akt. Additionally, we discuss the interplay between
H2O2-mediated inactivation of protein tyrosine phosphatases
(PTPs) and kinase activation on net cellular levels of tyrosine
(Tyr) phosphorylation. Key enzymes involved in the
generation and metabolism of H2O2 within EGFR signaling
pathways will also be covered. Finally, we focus on recent
examples from the literature demonstrating direct oxidation and
regulation of EGFR by H2O2 and how these discoveries may
form the basis for the development of new therapeutic
strategies for targeting this and other H2O2-modulated
pathways.
■
EARLY EVIDENCE OF EGF-MEDIATED H2O2
PRODUCTION AND REDOX REGULATION OF EGFR
SIGNALING
In 1995, Gamou and Shimizu published the first report to
suggest a connection between H2O2 and EGFR. In that study,
they examined the effect of exogenously added H2O2 on EGFR
phosphorylation.30 EGFR-hyperproducing human squamous
carcinoma NA cells treated with H2O2 (0−1 mM) exhibited an
increase in the level of incorporation of [32P]phosphate, albeit
at half the signal observed for EGF-stimulated cells. On the
basis of data obtained from tryptic phosphopeptide mapping,
this discrepancy was attributed to the fact that H2O2 might
preferentially enhance EGFR Tyr phosphorylation, whereas
EGF stimulation would trigger both serine/threonine (Ser/
Thr) and Tyr receptor phosphorylation.
During this same time frame (i.e., the mid-1990s), H2O2
molecules expanded beyond the traditional view as “toxic
byproducts of aerobic metabolism” and began to emerge as
secondary messengers in physiological cell signaling. For H2O2
to serve as a signaling molecule, its concentration must increase
rapidly above the steady-state threshold (i.e., high nanomolar to
low millimolar) and remain elevated long enough for it to
oxidize protein effectors.31 In one of the earliest examples,
platelet-derived growth factor (PDGF) was shown to induce
endogenous H2O2 generation, which is correlated with
enhanced Tyr phosphorylation, activation of MAPK pathways,
9955
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
(MAPK). Oxidative stress is known to influence the MAPK
signaling pathways, but little is known about the molecular
mechanisms responsible for such effects.37 For example, Erk1/2
is activated in response to exogenous H2O2, which enhances
cell survival after oxidant injury.38,39 On the other hand, it is not
known whether the activity of Erk1/2 kinase is directly
modulated by ROS.
EGFR also transmits signals through the PI3K/Akt pathway.40 In response to EGF stimulation, PI3K increases the
levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which
leads to recruitment and activation of serine/threonine kinase,
Akt at the plasma membrane.41,42 The activity of the PI3K/Akt
pathway is balanced by action of the opposing lipid
phosphatase, PTEN, which will be discussed in later sections
of this review. In cells overexpressing NADPH oxidase isoform
Nox1, an increased level of production of endogenous H2O2
elevates intracellular PIP3 levels and consequently abrogates the
ability of PTEN to promote downstream signaling.43 EGFRdependent activation of Akt has also been shown to enhance
cell survival during oxidative stress-induced apoptosis, analogous to Erk1/2.44 Interestingly, structural analysis by X-ray
crystallography has revealed that Akt2 can form an intramolecular disulfide bond between two cysteines in the
activation loop, which inhibits the kinase.45 Overexpression of
glutaredoxin (Grx) has been shown to protect Akt against
H2O2-induced oxidation, resulting in sustained phosphorylation
of Akt and inhibition of apoptosis.46 A more recent study is
indicative of isoform-specific regulation of Akt by PDGFinduced H2O2.47 In particular, the authors demonstrate that
Akt2 Cys124 undergoes sulfenic acid modification during
growth factor signaling, which inactivates the kinase.
EGFR activation can also occur through a process known as
transactivation. In this scenario, ligand-inaccessible RTKs may
still initiate downstream signaling in lieu of ligand−receptorinduced activation. Production of H2O2 induced by other
ligands such as angiotensin II activates c-Src, a redox-regulated
kinase.48 In turn, EGFR undergoes activation through c-Srcinitiated receptor Tyr phosphorylation to propagate downstream signaling.48−50 Neighboring EGFR kinases may then
undergo activation in a lateral-based mechanism. Transactivation represents another level by which EGFR activity
can be modulated by H2O2. In addition, other studies have
implicated oxidative inactivation of PTPs in promoting EGFR
transactivation.51
Figure 2. Oxidative modification of cysteine residues by hydrogen
peroxide (H2O2). (A) The initial reaction product of a thiolate with
H2O2 yields sulfenic acid (RSOH). This modification, also known as
sulfenylation, is reversible and can be directly reduced back to the thiol
form or indirectly through disulfide bond formation. (B) Sulfenic acids
can be stabilized by the protein microenvironment and/or undergo
subsequent modification. For example, they can condense with a
second cysteine in the same protein or a different protein to generate a
disulfide bond. Alternatively, reaction with the low-molecular weight
thiol glutathione (GSH, red circle) affords a mixed disulfide through a
process known as S-glutathionylation. In a few proteins, such as
PTP1B, nucleophilic attack of a backbone amide on RSOH results in
sulfenamide formation. Sulfenyl groups can also oxidize further to the
sulfinic (RSO2H) and/or sulfonic (RSO3H) acid form under
conditions of high oxidative stress.
activation may have separate functions and represent an
alternate mechanism by which EGFR signaling can be tuned
in parallel to treatment with its native ligand.
■
■
INTERPLAY OF REVERSIBLE TYROSINE
PHOSPHORYLATION AND REDOX-DEPENDENT
SIGNALING
Regulation of tyrosine phosphorylation depends on the delicate
balance between the action of protein kinases and phosphatase
(Figure 3B,C). In EGFR signaling, the coordinated regulation
of this equilibrium allows for rapid response to changing
growth factor levels, whereas dysregulated kinase and
phosphatase activity can have severe pathological consequences
such as cancer, diabetes, and inflammation.52,53 Because the
balance of these two opposing forces is a central event in cell
signaling, it is not surprising that phosphatase as well as kinase
activities are tightly regulated at several levels, including
cysteine-based redox modulation. In large part, because of
early studies by Denu and Tanner,54 attention rapidly focused
on PTPs as the direct targets of H2O2 (Figure 3D). The PTP
superfamily contains a signature motif, (I/V)HCXXGXXR(S/
T), which includes an invariant cysteine residue that functions
EFFECT OF H2O2 ON SIGNALING NETWORKS
DOWNSTREAM OF EGFR
After ligand binding, EGFR transmits activation signals to
prominent downstream cascades, including the Ras/MAPK and
PI3K/Akt pathways (Figure 3A). In addition to the overall
increase in the level of Tyr autophosphorylation of EGFR, the
endogenous generation of H2O2 also appears to modulate the
activity of these two signaling routes. Receptor activation
initiates the Ras-Raf-MEK-Erk1/2 signaling module through
recruitment of Src homology 2 domain-containing (SHC)
adaptor protein, growth factor receptor-bound protein 2
(Grb2), and a guanine nucleotide exchange protein (SOS) to
form the SHC−Grb2−SOS complex, a process stimulated by
elevated intracellular H2O2 levels.36 Once associated with the
receptor, SOS facilitates guanine nucleotide exchange to
activate Ras, which subsequently activates a kinase cascade
that includes Raf (MAP3K), MEK (MAP2K), and Erk1/2
9956
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
Figure 3. EGFR signaling pathways and general mechanisms for thiol-based redox modulation of signaling proteins. (A) Binding of EGF to EGFR
induces receptor dimerization, followed by autophosphorylation of tyrosine (Tyr) residues (red circles) within its cytoplasmic domain. In turn, these
phosphorylated Tyr residues serve as docking sites for associating proteins to activate a number of downstream signaling cascades. Two such
pathways, Ras/ERK and PI3K/AKT, are shown here for the sake of simplicity. The EGF−EGFR interaction also triggers the assembly and activation
of NADPH oxidase (Nox) complexes, followed by subsequent production of H2O2 through spontaneous dismutation or action of SOD. Once
formed, endogenous H2O2 may pass through specific aquaporin (AQP) channels and/or diffuse across the membrane to reach the intracellular
cytosol. Transient increases in H2O2 levels lead to the oxidation of local redox targets. (B) Model for redox-dependent signal transduction. Protein
tyrosine kinases (PTKs) catalyze the transfer of γ-phosphoryl groups from ATP to tyrosine hydroxyls of proteins, whereas protein tyrosine
phosphatases (PTPs) remove phosphate groups from phosphorylated tyrosine residues. PTPs function in a coordinated manner with PTKs to
control signaling pathways to regulate a diverse array of cellular processes. (C) Regulatory cysteines in protein kinases can undergo oxidation or
reduction to modulate their function. Depending on the kinase, redox modifications can stimulate or inhibit function. (D) Oxidation of the
conserved active site cysteine residue in PTPs inactivates these enzymes, which can be restored by reducing the oxidized residue to its thiol form.
SOx represents oxidized cysteine.
as a nucleophile in catalysis. The catalytic cysteine of PTPs is
characterized by a low pKa value ranging from 4.6 to 5.5
because of the unique electrostatic environment of the active
site, which also renders the enzyme susceptible to inactivation
by reversible oxidation.55,56 Second-order rate constants for
oxidation of the PTP cysteine thiolate by H2O2 have been
measured in vitro and range from 10 to 160 M−1 s−1.54,57,58
Protein tyrosine phosphatase 1B (PTP1B) functions as a
negative regulator of the EGFR signaling pathway by directly
targeting phosphorylated tyrosine residues that control signaling output.59−62 Given that PTP1B is localized exclusively to
the cytoplasmic face of the endoplasmic reticulum (ER), it has
been proposed to dephosphorylate activated EGFR at sites of
contact between the ER and plasma membranes or upon
trafficking of internalized EGFR in the proximity of the ER.63,64
Studies reported in 1998 provided the first indication that EGFmediated H2O2 production is correlated with oxidation and
inactivation of PTP1B.65 The level of incorporation of
radiolabeled iodoacetic acid (IAA), a sulfhydryl-modifying
reagent that reacts with active site Cys215 of PTP1B,54 was
decreased in PTP1B after EGF stimulation of A431 cells,
consistent with oxidation of this essential residue. Conversely,
treatment with dithiothreitol (DTT), thioredoxin (Trx), or
glutaredoxin (Grx) as a reductant readily reversed PTP1B
inhibition. Interestingly, assays with recombinant protein
indicated that the Trx system functioned more efficiently as
an electron donor for PTP1B reactivation, as compared to Grx
or glutathione (GSH), suggesting that Trx may function as the
physiological reductant.65
The phosphatase and tensin homologue (PTEN) is another
PTP known to maintain a closely intertwined relationship with
EGFR. PTEN exhibits dual protein and lipid phosphatase
activity and functions as a negative regulator of the PI3K/Akt
signaling pathway,66 one of the two major signaling routes
downstream of EGFR. PTEN contains five cysteine residues in
its catalytic domain and undergoes reversible inactivation by
H2O2.67 Site-directed mutagenesis and mass spectrometry
indicated that Cys124 was the primary target of H2O2, yielding
a sulfenic acid intermediate that condenses with Cys71 to form
an intramolecular disulfide bond.68 Cellular studies also point
to a connection between growth factor-induced generation of
H2O2 and reversible inactivation of PTEN. For example, EGF
stimulation of cells results in elevated levels of oxidized PTEN
in lysates, as indicated by an electrophoretic mobility shift assay
that reports on disulfide formation.69 In an alternative
approach, protein thiols are alkylated by N-ethylmaleimide
(NEM) in lysates generated from growth factor-stimulated
cells. Reversibly oxidized protein thiols are then reduced with
DTT and alkylated with biotin-conjugated maleimide. This
method was used to examine reversible oxidation of PTEN in
response to EGFR activation.69 Together, these studies indicate
that EGF-induced activation of PI3K correlates with
9957
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
inactivation of the opposing phosphatase, PTEN. In this way,
PTEN inactivation by endogenous H2O2 serves as a positive
feedback loop to enhance PIP3 accumulation and/or Akt
activation during EGFR signaling.
SHP2 and DEP-1 represent two additional phosphatases that
have been shown to interact with EGFR. SHP2 directly
associates with EGFR through its SH2 domains and regulates
interactions of the receptor with downstream signaling
components such as Ras.70 Colocalization of SHP2 with
EGFR occurs at low nanomolar concentrations (4 ng/mL) of
EGF, and SHP2 was identified as the most sensitive PTP in
response to signal-derived H2O2.71 It is unknown if DEP-1
colocalizes with EGFR intracellularly; however, evidence
implicates in vitro oxidation of DEP-1.72
Superoxide dismutase 1 (SOD1) is another enzyme involved
in redox mediation of growth factor signaling. SOD1 is an
abundant copper/zinc enzyme located in the cytoplasm and
belongs to the SOD family of enzymes. Other members of this
family include mitochondrial SOD2 (manganese) and extracellular SOD3 (copper/zinc). Although superoxide can spontaneously dismutate to form H2O2, the second-order rate
constant of the reaction is enhanced >10000-fold by
SOD.74,75 Indeed, SODs catalyze the conversion of superoxide
into H2O2 and molecular O2 to maintain superoxide at a low
steady-state concentration (∼10−10 M).83,84 Inhibition of SOD1
by the tetrathiomolybdate inhibitor, ATN-224, increases the
intracellular concentration of superoxide at the expense of
H2O2 production, thereby attenuating EGFR and Erk1/2
phosphorylation.85
Membrane transport represents another important mechanism for modulating endogenous H2O2 produced during EGFR
signaling. Early studies demonstrated that EGF stimulation of
cells leads to a rapid increase in the concentration of
extracellular H2O2 and that the addition of catalase to culture
medium was sufficient to inhibit EGFR autophosphorylation.86,87 These findings raise the question of how extracellularly generated H2O2 could mediate intracellular signaling
pathways. Although it had been largely assumed that H2O2
could diffuse across cellular membranes, more recent evidence
indicates that H2O2 may preferentially enter the cell through
specific plasma membrane aquaporin channels.88−90 Collectively, these studies suggest that the number or type of
aquaporins expressed on the cell surface might modulate the
level of intracellular H2O2 available for signaling.
Efforts have also focused on delineating mechanisms for
regulating intracellular H2O2 levels during EGF-mediated
signaling. In this regard, superoxide dismutases, catalase, and
other peroxidases all function to protect cells against undue
oxidative stress. Prxs are a family of thiol-based peroxidases that
catalyze the dismutation of H2O2 into water and molecular
oxygen.91 Prx possesses two cysteines that transiently oxidize to
form a disulfide as it metabolizes H2O2. The disulfide in Prx is
subsequently reduced by the protein disulfide reductase, Trx, to
complete the catalytic cycle. Overexpression of the PrxII
isoform reduces EGF-induced intracellular H2O2 levels.92
Another study demonstrated that PrxII overexpression is
associated with a decrease in the level of EGF-induced cellular
PIP3, whereas a dominant negative (DN) form of PrxII
increased PIP3 levels, presumably by H2O2 modulation of the
PTEN redox state.69 Similarly, overexpression of another
antioxidant enzyme, glutathione peroxidase-1 (Gpx1), decreased the level of Tyr phosphorylation of EGFR, activation
of Akt, and cellular proliferation.93 In addition to SODs and
peroxidases, cells rely on the glutaredoxin/glutathione (Grx/
GSH), Trx/thioredoxin reductase (Trx/TrxR), and glutathione/glutathione reductase (GSH/GSR) buffering systems.
For example, EGFR signaling is associated with subcellular
compartmental oxidation of Trx1. Jones and colleagues have
measured the redox states of cytosolic and nuclear Trx1 and
mitochondrial Trx2 using redox Western blot methods during
endogenous H2O2 production induced by EGF signaling.94
Interestingly, results from this study showed that only the
cytoplasmic Trx1 pool undergoes significant oxidation in
response to growth factor treatment. Furthermore, the GSH/
GSSG redox couple, which was also examined in this study, did
not undergo oxidation. This work suggests that physiological
H2O2 generation in response to EGF signaling is specifically
■
GENERATION AND METABOLISM OF H2O2
DURING EGFR SIGNALING
The family of NADPH oxidase (Nox) enzymes and their dual
oxidase counterparts (Duox) generate superoxide by transferring electrons from cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen.73 Once it is
generated, superoxide is dismutated spontaneously (∼105 M−1
s−1 at pH 7) or enzymatically by superoxide dismutase (SOD;
∼10 9 M−1 s−1) to H 2O2 and molecular O2 .74,75 The
prototypical Nox isoform, Nox2 (also known as gp91phox),
was originally identified and characterized in macrophages and
neutrophils, where it functions as an integral part of the innate
immune system.76 The active form of Nox2 exists as a
multisubunit complex, consisting of the membrane-bound
cytochrome b558 (gp91phox and p22phox), several cytosolic
proteins (p47phox, p40phox, and p67phox), and the small GTPase
Rac1. Since the initial discovery of Nox2, other Nox
homologues (Nox1−5 and Duox1 and -2) have been identified
in almost every cell type, localized both to the plasma
membrane (where they produce superoxide extracellularly)
and to intracellular organelles, and serve as major sources of
H2O2 for signaling.77−79
Several Nox isoforms play a critical role in EGFR-mediated
signaling cascades. For example, Park et al. demonstrated that
the PI3K pathway constitutes a positive feedback loop for Nox1
activation in growth factor-stimulated cells.43 This study
demonstrated that the Rac-guanine nucleotide exchange factor
(GEF) βPix is required for and also augments EGF-induced
generation of H2O2. In this loop, β-Pix and activated Rac1 bind
to the C-terminal region of Nox1, relieving autoinhibitory
constraints. Independent studies have also shown that
phosphorylation of Nox activator 1 (NOXA1) on Ser282 by
Erk1/2 kinases and on Ser172 by protein kinases C and A
weakens the binding of Rac1 to downregulate Nox1.80,81
Nox4 is an isoform that regulates the activity of internalized
EGFR. Keaney and colleagues report that Nox4 is localized to
the ER in vascular endothelial cells where it appears to regulate
the activity of PTP1B in a spatially dependent manner.82 Nox4dependent oxidation of PTP1B required colocalization of both
proteins in the ER, as shown by targeting PTP1B to the
cytoplasm. Importantly, the study also demonstrated that
Nox4-dependent oxidation and inactivation of PTP1B are
correlated with a reduced level of phosphorylation of EGFR in
the proximity of the ER. The significance of colocalization was
also verified by ER targeting of the antioxidant enzyme,
catalase. Lastly, EGF stimulation of A431 cells leads to the
formation of a specific complex between Nox2 and EGFR, as
demonstrated by co-immunoprecipitation experiments.71
9958
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
associated with oxidation of the Trx1 system and not the GSH
system. However, whether Trx1 oxidation is part of the
signaling mechanism itself or simply results from peroxidasedependent termination of the redox signal remains an active
area of investigation.
The estimated intracellular steady-state concentration of
H2O2 hovers in the low nanomolar to low micromolar range.31
Then again, these estimates assume that H2O2 is uniformly
distributed throughout the cell. Given that the source of H2O2
produced for EGF signaling (e.g., Nox enzymes) is localized to
specific regions of the cell, it stands to reason that signalmediated changes in H2O2 concentration may not be
homogeneous throughout the cell. Rather, the oxidant
concentration near a source of generation must achieve high
local concentrations to function effectively as a second
messenger. A growing body of research has focused on
delineating the mechanisms that facilitate localized increases
in intracellular H2O2 levels during growth factor signaling.
Although the cell contains millimolar concentrations of GSH, it
reacts too slowly with H2O2 to provide much buffering
capacity.95 By contrast, Prxs are extremely efficient at H2O2
elimination, reducing H2O2 with second-order rate constants of
105−108 M−1 s−1.96,97 Recent work reported by the Rhee
laboratory has shown that membrane-localized PrxI can be
deactivated by phosphorylation in EGF-stimulated cells and in
mice during wound healing.98 Knockdown experiments
suggested that c-Src kinase is at least partially responsible for
PrxI phosphorylation. RTK activation (e.g., PDGFR) can also
lead to overoxidation of the PrxII isoform catalytic cysteine to
sulfinic acid, resulting in a transiently inactivated protein.98
Collectively, these studies demonstrate that selective inactivation of PrxI and PrxII allows for transient H2O2 accumulation
around plasma membranes where signaling components are
concentrated, while simultaneously preventing toxic accumulation of ROS elsewhere in the cell during EGF signaling
(Figure 4). Other mechanisms for modulating localized redox
buffering capacity surely await discovery.
Figure 4. Redox regulation of peroxiredoxins (Prxs) during EGFR
signaling. Receptor activation results in localized phosphorylation and
inactivation of peroxiredoxin I (PrxI) by PTKs, such as the redoxregulated cytoplasmic Src (c-Src). Deactivation of PrxI diminishes the
redox buffering capacity adjacent to the cell membrane, allowing for a
transient and localized increase in H2O2 levels for signal transduction.
Additionally, elevated H2O2 concentrations can inactivate Prx2 by
oxidation of its catalytic cysteine to sulfinic acid.
we have recently described a new technology that allows for the
detection and visualization of sulfenylation proteins within
intact cells,103−106 thereby circumventing concerns associated
with the analysis of lysates and/or homogenates, including
limited spatiotemporal resolution and oxidation artifacts
inherent to the lysis procedure.107 Inspired by earlier work
demonstrating the selective reaction of 5,5-dimethyl-1,3cyclohexanedione (commercially known as dimedone) with
protein sulfenic acids108 (Figure 5A), we developed a suite of
bifunctional probes that contain a membrane-permeable
analogue of dimedone coupled to an azide or alkyne chemical
handle (Figure 5B). An orthogonally functionalized biotin or
fluorescent tag can be appended post-cell labeling for detection
via the Staudinger ligation109 or Huisgen [3+2] cycloaddition
also known as click chemistry.110 Overall, this general approach
provides a facile method for monitoring protein sulfenylation
directly in living cells (Figure 5C).
Employing this method, we demonstrated that EGFR
undergoes sulfenylation in response to addition of EGF, even
at low nanomolar concentrations of growth factor71 (Figure
6A). Reciprocal immunoprecipitation analysis also showed that
EGFR and Nox2 became associated in an EGF-dependent
fashion.71 The intracellular kinase domain of EGFR contains six
cysteine residues, one of which (Cys797) is located in the ATPbinding pocket (Figure 6B) and is conserved among nine
additional receptor and nonreceptor tyrosine kinases (Figure
6C).111 Given its active site location and conservation, we
hypothesized that Cys797 might be preferentially targeted by
endogenous H2O2. Of particular relevance, this residue is
selectively targeted by irreversible EGFR inhibitors, such as
afatinib, extensively used in basic research and clinical trials for
breast and non-small-cell lung cancers.112,113 Consistent with
this proposal, pretreatment of cells with inhibitors that
irreversibly modify Cys797 prevented sulfenylation of EGFR.
Mass spectrometry was subsequently used to verify Cys797 as
■
REGULATION OF INTRINSIC EGFR TYROSINE
KINASE ACTIVITY THROUGH CYSTEINE
OXIDATION
As outlined above, a growing body of evidence demonstrates
that EGFR activity and downstream signaling pathways are
regulated by redox-based mechanisms. Up until this point, we
have only considered downstream events that enhance the
overall extent of EGFR activation, such as PTP inactivation. We
have also highlighted key regulatory themes in redox signaling,
including colocalization of sources and/or targets of H2O2 and
modulation of local redox buffering capacity. Until recently,
there was scant evidence to indicate that the intrinsic tyrosine
kinase activity of EGFR itself might be regulated by
endogenous H2O2 produced during cell signaling.99−101 In
the section below, we recount recent work from our own
laboratory, which has demonstrated that EGFR is directly
modulated by endogenous H2O2, as well as studies from other
groups that hint at the possibility of modification by reactive
nitrogen species (RNS).
Previous data from our group demonstrated that breast
cancer cells associated with an increased level of expression of
EGFR are correlated with a substantial increase in the level of
protein sulfenylation.102 Prompted by this observation, we
conducted a more detailed investigation of EGF-induced
protein sulfenylation in A431 cells.71 Facilitating these studies,
9959
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
Figure 5. General strategy for detecting protein cysteine sulfenylation (RSOH) in cells. (A) Chemoselective reaction between sulfenic acid and 5,5dimethyl-1,3-cyclohexanedione (dimedone, 1). (B) Azide- and alkyne-functionalized small-molecule probes for trapping and tagging protein sulfenic
acids include DAz-2 (2) and DYn-2 (3). (C) Detection of protein sulfenic acids in living cells. Target cells are incubated with cell-permeable probes
to trap and tag protein sulfenic acids in situ. In subsequent steps, lysates are prepared and tagged proteins are further elaborated by attachment of
biotin or fluorescence labels via click chemistry that permits detection by Western blot or in-gel fluorescence. Alternatively, biotinylated proteins may
be enriched for proteomic analysis.
Figure 6. Model for H2O2-dependent regulation of EGFR tyrosine kinase activity. (A) Binding of EGF induces production of H2O2 through Nox2.
Nox-derived H2O2 directly modifies EGFR cysteine (Cys797) to sulfenic acid in the active site, which enhances its tyrosine kinase activity.
Endogenous H2O2 can also oxidize and deactivate localized PTPs, leading to a net increase in the level of EGFR phosphorylation. (B) Crystal
structure of the EGFR kinase domain (Protein Data Bank entry 3GT8) bound to AMP-PNP, a hydrolysis resistant ATP analogue, and Mg2+. Dashed
yellow lines and accompanying numbers indicate the distance (angstroms) between the γ-sulfur atom of Cys797 and key substrate functional groups.
Note also that Cys797 can adopt different rotamers and sulfenylation of this residue may enhance its ability to participate in electrostatic and
hydrogen bonding interactions with its substrate. (C) Abbreviated sequence alignment of EGFR and nine other kinases that harbor a cysteine at the
structural position that corresponds to Cys797 (adapted from ref 112).
the specific site of oxidation. Finally, oxidation of Cys797
increased EGFR kinase activity by approximately 5-fold. To put
these findings into context, a comparable degree of stimulation
is observed for L858R and T790M oncogenic EGFR
mutations.114−116 Beyond the A431 cell line model, recent
studies by our group also indicate that wild-type and several
activated mutant EGFR kinases undergo sulfenylation in both
lung and breast tumors (T. H. Truong and K. S. Carroll,
unpublished data). Hence, it appears that oxidation of specific
residues in PTPs (catalytic Cys) and EGFR (Cys797) both
contribute to an increase in the level of downstream signaling
(Figure 6A). Current work is directed toward understanding
the molecular mechanism by which sulfenylation of EGFR
Cys797 enhances kinase activity. The proximity of Cys797 to
9960
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
Figure 7. Covalent cysteine-based protein targeting strategies. (A) Conventional covalent inhibitors of kinases inactivate their target through
covalent attachment to the cysteine thiol functional group. However, the electrophilic center (e.g., acrylamide, haloacetamide, and vinyl sulfonamide)
that reacts with the cysteine can exhibit nonspecific reactivity toward other cellular thiols, including glutathione present at millimolar concentrations
inside mammalian cells. The electrophile may also react with other nucleophilic functionalities present in biological systems (amino and imidazole
groups of amino acids, various reactive sites in nucleic acid bases, and water). (B) Orthogonal strategy as one potential mechanism for addressing
issues associated with employing an electrophilic functional group to target one cysteine among a sea of biological nucleophiles. According to this
approach, active site-directed small-molecule inhibitors containing a reactive nucleophilic substituent form a covalent bond with a sulfenic acidmodified cysteine side chain. Such modifications form transiently in specific proteins during H2O2-mediated signal transduction in normal cells but
form constitutively in diseases associated with chronically elevated levels of H2O2, including cancer. In the sulfenic acid oxidation state, the electrondeficient sulfur exhibits enhanced electrophilic character that can be selectively targeted by certain nucleophilic compounds. Because sulfenic acid is a
unique chemical moiety in biochemistry, this strategy might decrease the potential for off-target activity while retaining the advantages gained by
covalent targeting.
the ATP ligand as well as the C-helix and activation segment
raises the possibility of transition-state stabilization and/or
destabilization of its autoinhibited conformation.116
We have also applied our chemical biology approach to
monitor global changes in EGF-dependent protein sulfenylation.71 Interestingly, addition of growth factor led to widespread changes in protein sulfenylation within cells. EGF
induced cysteine oxidation in a dose- and time-dependent
manner, which was accompanied by concomitant fluxes in
intracellular H2O2 levels. Additional experiments showed that
treatment with cell-permeable PEGylated catalase attenuated
EGF-associated changes in protein sulfenylation, underscoring
the importance of endogenous H2O2. Although sulfenylation of
EGFR (and several PTPs) was the focus our recent study,71
many protein targets of EGF-induced endogenous H2O2
remain to be identified and will necessitate large-scale
proteomic analysis.
Remarkably, a number of studies indicate that EGFR may
also undergo modulation by RNS at cysteine residues distinct
from Cys797. Reaction of cysteine thiols with RNS, such as
nitric oxide (NO), generates nitrosothiols (S-NO). This
process, known as S-nitrosylation, is a well-established
reversible post-translational cysteine modification and has
been implicated in proliferative and antiproliferative cellular
effects.117 The approach most often used to identify protein
nitrosothiols is known as the biotin switch technique (BST).118
The BST is an indirect method whose success relies heavily on
the alkylation of free thiols and the selectivity and/or efficiency
of the ascorbate reducing agent toward nitrosothiols.119,120
Nonetheless, this method was utilized in two separate studies
reporting S-nitrosylation of EGFR. Treatment of several cell
types with an exogenous nitric oxide (NO) donor 1,1-diethyl-2hydroxy-2-nitrosohydrazine (DEA-NO) at a concentration of 1
mM inhibited EGFR autophosphorylation.121,122 Mutation of
Cys166 of EGFR, located in the extracellular domain, to serine
rendered the receptor resistant to NO-induced inhibition.
Alternatively, another cysteine residue located at the extracellular EGFR ligand-binding interface undergoes nitrosylation
after exposure to 1 mM exogenous NO donor S-nitroso-Lcysteine (Cys-NO).123 S-Nitrosylation of these residues may
inhibit EGFR-mediated signaling by interfering with the ligand
interaction site, although this proposal awaits evaluation. By
contrast, a more recent study demonstrates that S-nitrosylation
induced by the chemical NO donor (Z)-1-[N-(2-aminoethyl)N-(2-ammonioethyl)amino]diazen-1-ium 1,2-diolate known as
DETA-NO can upregulate EGFR signaling and correlates with
a transformed breast cancer phenotype.124,125 An important
goal for future research is to determine whether EGFR
undergoes direct S-nitrosylation in response to physiological
stimuli.
■
FUTURE PERSPECTIVES
Of the ∼95 receptor and nonreceptor PTKs in the human
genome, nine additional members harbor a cysteine residue
that is structurally homologous to EGFR Cys797 (Figure 6C),
including two EGFR subfamily members, HER2 and HER4.
Although this conclusion is speculative at this time, it is possible
that this group of kinases is regulated by oxidation of this
9961
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
residue. EGFR Cys797 and its structural analogues are located
at the N-terminal end of an α helix, also known as the Ncap
position. Interestingly, cysteine is the most sparsely occurring
Ncap residue in natural proteins, comprising <1% of all these
positions.126 Interaction of a cysteine located at the Ncap
position with the helix dipole can drastically lower the thiol
pKa and increases its reactivity.127,128 Of note, the Ncap effect
has also been attributed to the reactivity of the human PrxI
catalytic cysteine.98 Another subfamily of PTKs, which includes
cytoplasmic Src and FGFR1, contains a cysteine located within
a glycine rich loop that interacts with the γ-phosphate of
ATP.129 Oxidation of this residue inhibits the kinase activity of
c-Src and FGFR1 in vitro; however, neither kinase has been
confirmed as a direct target of endogenous H2O2 in cells. More
than 150 kinases have a cysteine in or around the nucleotidebinding site, some of which may play similar regulatory roles.
However, much more work will be required to define the scope
and molecular details underlying the redox-regulated kinome.
EGFR is mutated or amplified in a number of human
carcinomas, including breast and lung cancers, which has
motivated the development of selective kinase inhibitors,
including analogues that covalently modify Cys797 and are
currently in phase II and III clinical trials.130 The recent
findings that EGFR Cys797 undergoes sulfenic acid modification71 and that elevated EGFR and HER2 levels in cancer
cells correlate with a substantial increase in the extend of global
protein sulfenylation102 raise several fundamental questions visà-vis cysteine oxidation and thiol-targeted irreversible inhibitors. For example, the acrylamide moiety of irreversible EGFR
inhibitors undergoes Michael addition with Cys797 in its thiol
form, but these inhibitors would not react with the sulfenic acid
or other cysteine chemotypes (Figure 7A). Thus, oxidation of
Cys797 could affect the potency of these inhibitors, particularly
under conditions of oxidative stress often associated with
cancer. On the other hand, the sulfenic acid moiety represents
an entirely new opportunity in covalent inhibitor design
whereby the electrophilic S atom is targeted using a
nucleophilic warhead (Figure 7B). In this approach, the
propensity for specific cysteine residues in kinases, and other
therapeutically important proteins, to undergo sulfenylation
could be exploited for the development of inhibitors that target
this unique modification, similar to the proof-of-concept
compounds we have recently reported to target oxidized
PTPs.131
may target distinct cysteine residues in the same protein and
thus lead to unique regulatory outcomes. Given that aberrant
sulfenylation of proteins is linked to aggressive cancer
phenotypes and that genetic lesions in H2O2-metabolizing
enzymes can contribute to tumorigenesis, defining mechanisms
that control reversible protein sulfenylation will be vital to
understanding both human physiology and disease. Finally, it is
hoped that the discovery of EGFR as a direct target of H2O2
will lead to a broader examination of the redox-regulated
kinome and the development of an orthogonal nucleophilic
strategy for the covalent inhibition of therapeutically important
proteins.
CONCLUSIONS
The perspectives presented here highlight the emerging and
rapidly expanding role of redox regulation during EGFR
signaling. These studies point to the unique chemistry of
reactive cysteine residues within specific target proteins,
including EGFR. These redox reactions allow covalent
regulation of protein function, much like phosphorylation.
The expanding array of modifications that target cysteine
suggests that we are just beginning to understand the molecular
basis for the specificity of redox signaling. One theme that has
consistently emerged in numerous studies is the colocalization
of the oxidant sources with the redox-regulated target protein.
As a case in point, ligand activation triggers the association of
EGFR and the NADPH oxidase, Nox2. We have also tried to
emphasize the value of selective and cell-permeable chemical
approaches to elucidating regulatory mechanisms that govern
H2O2-mediated sulfenylation of proteins. In addition, we are
just beginning to appreciate that different biological oxidants
(1) Schlessinger, J. (2000) Cell signaling by receptor tyrosine kinases.
Cell 103, 211−225.
(2) Schlessinger, J. (2002) Ligand-induced, receptor-mediated
dimerization and activation of EGF receptor. Cell 110, 669−672.
(3) Cohen, S. (1962) Isolation of a mouse submaxillary gland protein
accelerating incisor eruption and eyelid opening in the new-born
animal. J. Biol. Chem. 237, 1555−1562.
(4) Cohen, S., Carpenter, G., and King, L., Jr. (1980) Epidermal
growth factor-receptor-protein kinase interactions. Co-purification of
receptor and epidermal growth factor-enhanced phosphorylation
activity. J. Biol. Chem. 255, 4834−4842.
(5) Carpenter, G., King, L., Jr., and Cohen, S. (1978) Epidermal
growth factor stimulates phosphorylation in membrane preparations in
vitro. Nature 276, 409−410.
(6) Cohen, S. (2008) Origins of growth factors: NGF and EGF. J.
Biol. Chem. 283, 33793−33797.
(7) Macias, A., Azavedo, E., Hagerstrom, T., Klintenberg, C., Perez,
R., and Skoog, L. (1987) Prognostic significance of the receptor for
epidermal growth factor in human mammary carcinomas. Anticancer
Res. 7, 459−464.
■
AUTHOR INFORMATION
Corresponding Author
*The Scripps Research Institute, Scripps Florida, 130 Scripps
Way, Jupiter, FL 33458. E-mail: kcarroll@scripps.edu. Phone:
(561) 228-2460. Fax: (561) 228-2919.
Funding
This work was supported by National Institutes of Health
Grant GM102187 (K.S.C.) and funding from the Camille
Henry Dreyfus Teacher Scholar Award (K.S.C.).
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS
RTK, receptor tyrosine kinase; PTK, protein tyrosine kinase;
PTP, protein tyrosine phosphatase; EGF, epidermal growth
factor; EGFR, EGF receptor; MAPK, mitogen-activated protein
kinase; PI3K, phosphatidylinositol 3′-kinase; PTEN, phosphatase and tensin homologue; PIP3, phosphatidylinositol 3,4,5triphosphate; H2O2, hydrogen peroxide; ROS, reactive oxygen
species; Nox, NADPH oxidase; NADPH, nicotinamide adenine
dinucleotide phosphate; SOD, superoxide dismutase; Prx,
peroxiredoxin; Gpx, glutathione peroxidase; Grx, glutaredoxin;
GSH, glutathione; GSR, glutathione reductase; Trx, thioredoxin; TrxR, thioredoxin reductase; ER, endoplasmic reticulum;
DTT, dithiothreitol; IAA, iodoacetic acid; IAM, iodoacetamide;
NEM, N-ethylmaleimide; DCFH-DA, 2′,7′-dichlorofluorescein
diacetate; DN, dominant-negative; RNS, reactive nitrogen
species; NO, nitric oxide.
■
■
9962
REFERENCES
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
(8) Yarden, Y., and Sliwkowski, M. X. (2001) Untangling the ErbB
signalling network. Nat. Rev. Mol. Cell Biol 2, 127−137.
(9) Herbst, R. S. (2004) Review of epidermal growth factor receptor
biology. Int. J. Radiat. Oncol. Biol. Phys. 59, 21−26.
(10) Bae, Y. S., Kang, S. W., Seo, M. S., Baines, I. C., Tekle, E.,
Chock, P. B., and Rhee, S. G. (1997) Epidermal growth factor (EGF)induced generation of hydrogen peroxide. Role in EGF receptormediated tyrosine phosphorylation. J. Biol. Chem. 272, 217−221.
(11) Miller, E. W., Tulyathan, O., Isacoff, E. Y., and Chang, C. J.
(2007) Molecular imaging of hydrogen peroxide produced for cell
signaling. Nat. Chem. Biol. 3, 263−267.
(12) Lo, Y. Y., and Cruz, T. F. (1995) Involvement of reactive oxygen
species in cytokine and growth factor induction of c-fos expression in
chondrocytes. J. Biol. Chem. 270, 11727−11730.
(13) Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., and Finkel, T.
(1995) Requirement for generation of H2O2 for platelet-derived
growth factor signal transduction. Science 270, 296−299.
(14) Colavitti, R., Pani, G., Bedogni, B., Anzevino, R., Borrello, S.,
Waltenberger, J., and Galeotti, T. (2002) Reactive oxygen species as
downstream mediators of angiogenic signaling by vascular endothelial
growth factor receptor-2/KDR. J. Biol. Chem. 277, 3101−3108.
(15) May, J. M., and de Haen, C. (1979) Insulin-stimulated
intracellular hydrogen peroxide production in rat epididymal fat
cells. J. Biol. Chem. 254, 2214−2220.
(16) Meier, B., Radeke, H. H., Selle, S., Younes, M., Sies, H., Resch,
K., and Habermehl, G. G. (1989) Human fibroblasts release reactive
oxygen species in response to interleukin-1 or tumour necrosis factorα. Biochem. J. 263, 539−545.
(17) Griendling, K. K., Minieri, C. A., Ollerenshaw, J. D., and
Alexander, R. W. (1994) Angiotensin II stimulates NADH and
NADPH oxidase activity in cultured vascular smooth muscle cells. Circ.
Res. 74, 1141−1148.
(18) Rhee, S. G. (2006) Cell signaling. H2O2, a necessary evil for cell
signaling. Science 312, 1882−1883.
(19) D’Autreaux, B., and Toledano, M. B. (2007) ROS as signalling
molecules: Mechanisms that generate specificity in ROS homeostasis.
Nat. Rev. Mol. Cell Biol. 8, 813−824.
(20) Dickinson, B. C., and Chang, C. J. (2011) Chemistry and
biology of reactive oxygen species in signaling or stress responses. Nat.
Chem. Biol. 7, 504−511.
(21) Finkel, T. (2011) Signal transduction by reactive oxygen species.
J. Cell Biol. 194, 7−15.
(22) Reddie, K. G., and Carroll, K. S. (2008) Expanding the
functional diversity of proteins through cysteine oxidation. Curr. Opin.
Chem. Biol. 12, 746−754.
(23) Paulsen, C. E., and Carroll, K. S. (2010) Orchestrating redox
signaling networks through regulatory cysteine switches. ACS Chem.
Biol. 5, 47−62.
(24) Jacob, C., Battaglia, E., Burkholz, T., Peng, D., Bagrel, D., and
Montenarh, M. (2012) Control of oxidative posttranslational cysteine
modifications: From intricate chemistry to widespread biological and
medical applications. Chem. Res. Toxicol. 25, 588−604.
(25) Zheng, M., Aslund, F., and Storz, G. (1998) Activation of the
OxyR transcription factor by reversible disulfide bond formation.
Science 279, 1718−1721.
(26) Chen, C. Y., Willard, D., and Rudolph, J. (2009) Redox
regulation of SH2-domain-containing protein tyrosine phosphatases by
two backdoor cysteines. Biochemistry 48, 1399−1409.
(27) Aslan, M., and Ozben, T. (2003) Oxidants in receptor tyrosine
kinase signal transduction pathways. Antioxid. Redox Signaling 5, 781−
788.
(28) Tonks, N. K. (2005) Redox redux: Revisiting PTPs and the
control of cell signaling. Cell 121, 667−670.
(29) Finkel, T. (2012) From sulfenylation to sulfhydration: What a
thiolate needs to tolerate. Sci. Signaling 5, pe10.
(30) Gamou, S., and Shimizu, N. (1995) Hydrogen peroxide
preferentially enhances the tyrosine phosphorylation of epidermal
growth factor receptor. FEBS Lett. 357, 161−164.
(31) Stone, J. R., and Yang, S. (2006) Hydrogen peroxide: A
signaling messenger. Antioxid. Redox Signaling 8, 243−270.
(32) Bass, D. A., Parce, J. W., Dechatelet, L. R., Szejda, P., Seeds, M.
C., and Thomas, M. (1983) Flow cytometric studies of oxidative
product formation by neutrophils: A graded response to membrane
stimulation. J. Immunol. 130, 1910−1917.
(33) Giorgio, M., Trinei, M., Migliaccio, E., and Pelicci, P. G. (2007)
Hydrogen peroxide: A metabolic by-product or a common mediator of
ageing signals? Nat. Rev. Mol. Cell Biol. 8, 722−728.
(34) Halliwell, B., and Gutteridge, J. M. C. (1999) Free Radicals in
Biology and Medicine, Oxford University Press, Oxford, U.K.
(35) Goldkorn, T., Balaban, N., Matsukuma, K., Chea, V., Gould, R.,
Last, J., Chan, C., and Chavez, C. (1998) EGF-Receptor
phosphorylation and signaling are targeted by H2O2 redox stress.
Am. J. Respir. Cell Mol. Biol. 19, 786−798.
(36) Rao, G. N. (1996) Hydrogen peroxide induces complex
formation of SHC-Grb2-SOS with receptor tyrosine kinase and
activates Ras and extracellular signal-regulated protein kinases group of
mitogen-activated protein kinases. Oncogene 13, 713−719.
(37) McCubrey, J. A., Lahair, M. M., and Franklin, R. A. (2006)
Reactive oxygen species-induced activation of the MAP kinase
signaling pathways. Antioxid. Redox Signaling 8, 1775−1789.
(38) Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J.
(1996) Activation of mitogen-activated protein kinase by H2O2. Role
in cell survival following oxidant injury. J. Biol. Chem. 271, 4138−4142.
(39) Wang, X., Martindale, J. L., Liu, Y., and Holbrook, N. J. (1998)
The cellular response to oxidative stress: Influences of mitogenactivated protein kinase signalling pathways on cell survival. Biochem. J.
333 (Part 2), 291−300.
(40) Leslie, N. R. (2006) The redox regulation of PI 3-kinasedependent signaling. Antioxid. Redox Signaling 8, 1765−1774.
(41) Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese,
C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T.
(1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the
activation of protein kinase B. Science 277, 567−570.
(42) Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A.
(1997) Direct regulation of the Akt proto-oncogene product by
phosphatidylinositol-3,4-bisphosphate. Science 275, 665−668.
(43) Park, H. S., Lee, S. H., Park, D., Lee, J. S., Ryu, S. H., Lee, W. J.,
Rhee, S. G., and Bae, Y. S. (2004) Sequential activation of
phosphatidylinositol 3-kinase, βPix, Rac1, and Nox1 in growth
factor-induced production of H2O2. Mol. Cell. Biol. 24, 4384−4394.
(44) Wang, X., McCullough, K. D., Franke, T. F., and Holbrook, N. J.
(2000) Epidermal growth factor receptor-dependent Akt activation by
oxidative stress enhances cell survival. J. Biol. Chem. 275, 14624−
14631.
(45) Huang, X., Begley, M., Morgenstern, K. A., Gu, Y., Rose, P.,
Zhao, H., and Zhu, X. (2003) Crystal structure of an inactive Akt2
kinase domain. Structure 11, 21−30.
(46) Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K.,
and Kondo, T. (2003) Glutaredoxin exerts an antiapoptotic effect by
regulating the redox state of Akt. J. Biol. Chem. 278, 50226−50233.
(47) Wani, R., Qian, J., Yin, L., Bechtold, E., King, S. B., Poole, L. B.,
Paek, E., Tsang, A. W., and Furdui, C. M. (2011) Isoform-specific
regulation of Akt by PDGF-induced reactive oxygen species. Proc. Natl.
Acad. Sci. U.S.A. 108, 10550−10555.
(48) Ushio-Fukai, M., Griendling, K. K., Becker, P. L., Hilenski, L.,
Halleran, S., and Alexander, R. W. (2001) Epidermal growth factor
receptor transactivation by angiotensin II requires reactive oxygen
species in vascular smooth muscle cells. Arterioscler., Thromb., Vasc.
Biol. 21, 489−495.
(49) Chen, K., Vita, J. A., Berk, B. C., and Keaney, J. F., Jr. (2001) cJun N-terminal kinase activation by hydrogen peroxide in endothelial
cells involves SRC-dependent epidermal growth factor receptor
transactivation. J. Biol. Chem. 276, 16045−16050.
(50) Giannoni, E., Buricchi, F., Grimaldi, G., Parri, M., Cialdai, F.,
Taddei, M. L., Raugei, G., Ramponi, G., and Chiarugi, P. (2008) Redox
regulation of anoikis: Reactive oxygen species as essential mediators of
cell survival. Cell Death Differ. 15, 867−878.
9963
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
sulfenylation of the EGFR catalytic site enhances kinase activity. Nat.
Chem. Biol. 8, 57−64.
(72) Persson, C., Kappert, K., Engstrom, U., Ostman, A., and
Sjoblom, T. (2005) An antibody-based method for monitoring in vivo
oxidation of protein tyrosine phosphatases. Methods 35, 37−43.
(73) Lambeth, J. D. (2004) NOX enzymes and the biology of
reactive oxygen. Nat. Rev. Immunol. 4, 181−189.
(74) McCord, J. M., and Fridovich, I. (1969) Superoxide dismutase.
An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem.
244, 6049−6055.
(75) Hsu, J. L., Hsieh, Y., Tu, C., O’Connor, D., Nick, H. S., and
Silverman, D. N. (1996) Catalytic properties of human manganese
superoxide dismutase. J. Biol. Chem. 271, 17687−17691.
(76) Babior, B. M., Lambeth, J. D., and Nauseef, W. (2002) The
neutrophil NADPH oxidase. Arch. Biochem. Biophys. 397, 342−344.
(77) Suh, Y. A., Arnold, R. S., Lassegue, B., Shi, J., Xu, X., Sorescu, D.,
Chung, A. B., Griendling, K. K., and Lambeth, J. D. (1999) Cell
transformation by the superoxide-generating oxidase Mox1. Nature
401, 79−82.
(78) Cheng, G., Cao, Z., Xu, X., van Meir, E. G., and Lambeth, J. D.
(2001) Homologs of gp91phox: Cloning and tissue expression of
Nox3, Nox4, and Nox5. Gene 269, 131−140.
(79) De Deken, X., Wang, D., Many, M. C., Costagliola, S., Libert, F.,
Vassart, G., Dumont, J. E., and Miot, F. (2000) Cloning of two human
thyroid cDNAs encoding new members of the NADPH oxidase family.
J. Biol. Chem. 275, 23227−23233.
(80) Oh, H., Jung, H. Y., Kim, J., and Bae, Y. S. (2010)
Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk
desensitizes EGF-induced ROS generation. Biochem. Biophys. Res.
Commun. 394, 691−696.
(81) Kroviarski, Y., Debbabi, M., Bachoual, R., Perianin, A.,
Gougerot-Pocidalo, M. A., El-Benna, J., and Dang, P. M. (2010)
Phosphorylation of NADPH oxidase activator 1 (NOXA1) on serine
282 by MAP kinases and on serine 172 by protein kinase C and
protein kinase A prevents NOX1 hyperactivation. FASEB J. 24, 2077−
2092.
(82) Chen, K., Kirber, M. T., Xiao, H., Yang, Y., and Keaney, J. F., Jr.
(2008) Regulation of ROS signal transduction by NADPH oxidase 4
localization. J. Cell Biol. 181, 1129−1139.
(83) Fridovich, I. (1995) Superoxide radical and superoxide
dismutases. Annu. Rev. Biochem. 64, 97−112.
(84) Gardner, P. R., Raineri, I., Epstein, L. B., and White, C. W.
(1995) Superoxide radical and iron modulate aconitase activity in
mammalian cells. J. Biol. Chem. 270, 13399−13405.
(85) Juarez, J. C., Manuia, M., Burnett, M. E., Betancourt, O., Boivin,
B., Shaw, D. E., Tonks, N. K., Mazar, A. P., and Donate, F. (2008)
Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated
oxidation and inactivation of phosphatases in growth factor signaling.
Proc. Natl. Acad. Sci. U.S.A. 105, 7147−7152.
(86) DeYulia, G. J., Jr., Carcamo, J. M., Borquez-Ojeda, O., Shelton,
C. C., and Golde, D. W. (2005) Hydrogen peroxide generated
extracellularly by receptor-ligand interaction facilitates cell signaling.
Proc. Natl. Acad. Sci. U.S.A. 102, 5044−5049.
(87) DeYulia, G. J., Jr., and Carcamo, J. M. (2005) EGF receptorligand interaction generates extracellular hydrogen peroxide that
inhibits EGFR-associated protein tyrosine phosphatases. Biochem.
Biophys. Res. Commun. 334, 38−42.
(88) Bienert, G. P., Schjoerring, J. K., and Jahn, T. P. (2006)
Membrane transport of hydrogen peroxide. Biochim. Biophys. Acta
1758, 994−1003.
(89) Bienert, G. P., Moller, A. L., Kristiansen, K. A., Schulz, A.,
Moller, I. M., Schjoerring, J. K., and Jahn, T. P. (2007) Specific
aquaporins facilitate the diffusion of hydrogen peroxide across
membranes. J. Biol. Chem. 282, 1183−1192.
(90) Miller, E. W., Dickinson, B. C., and Chang, C. J. (2010)
Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling. Proc. Natl. Acad. Sci. U.S.A. 107, 15681−
15686.
(51) Reynolds, A. R., Tischer, C., Verveer, P. J., Rocks, O., and
Bastiaens, P. I. (2003) EGFR activation coupled to inhibition of
tyrosine phosphatases causes lateral signal propagation. Nat. Cell Biol.
5, 447−453.
(52) Tonks, N. K. (2006) Protein tyrosine phosphatases: From
genes, to function, to disease. Nat. Rev. Mol. Cell Biol. 7, 833−846.
(53) Blume-Jensen, P., and Hunter, T. (2001) Oncogenic kinase
signalling. Nature 411, 355−365.
(54) Denu, J. M., and Tanner, K. G. (1998) Specific and reversible
inactivation of protein tyrosine phosphatases by hydrogen peroxide:
Evidence for a sulfenic acid intermediate and implications for redox
regulation. Biochemistry 37, 5633−5642.
(55) Zhang, Z. Y., and Dixon, J. E. (1993) Active site labeling of the
Yersinia protein tyrosine phosphatase: The determination of the pKa
of the active site cysteine and the function of the conserved histidine
402. Biochemistry 32, 9340−9345.
(56) Lohse, D. L., Denu, J. M., Santoro, N., and Dixon, J. E. (1997)
Roles of aspartic acid-181 and serine-222 in intermediate formation
and hydrolysis of the mammalian protein-tyrosine-phosphatase PTP1.
Biochemistry 36, 4568−4575.
(57) Winterbourn, C. C., and Metodiewa, D. (1999) Reactivity of
biologically important thiol compounds with superoxide and hydrogen
peroxide. Free Radical Biol. Med. 27, 322−328.
(58) Sohn, J., and Rudolph, J. (2003) Catalytic and chemical
competence of regulation of cdc25 phosphatase by oxidation/
reduction. Biochemistry 42, 10060−10070.
(59) Flint, A. J., Tiganis, T., Barford, D., and Tonks, N. K. (1997)
Development of “substrate-trapping” mutants to identify physiological
substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. U.S.A.
94, 1680−1685.
(60) Liu, F., and Chernoff, J. (1997) Protein tyrosine phosphatase 1B
interacts with and is tyrosine phosphorylated by the epidermal growth
factor receptor. Biochem. J. 327 (Part 1), 139−145.
(61) Wu, Y., Kwon, K. S., and Rhee, S. G. (1998) Probing cellular
protein targets of H2O2 with fluorescein-conjugated iodoacetamide
and antibodies to fluorescein. FEBS Lett. 440, 111−115.
(62) Lou, Y. W., Chen, Y. Y., Hsu, S. F., Chen, R. K., Lee, C. L.,
Khoo, K. H., Tonks, N. K., and Meng, T. C. (2008) Redox regulation
of the protein tyrosine phosphatase PTP1B in cancer cells. FEBS J.
275, 69−88.
(63) Haj, F. G., Markova, B., Klaman, L. D., Bohmer, F. D., and Neel,
B. G. (2003) Regulation of receptor tyrosine kinase signaling by
protein tyrosine phosphatase-1B. J. Biol. Chem. 278, 739−744.
(64) Haj, F. G., Verveer, P. J., Squire, A., Neel, B. G., and Bastiaens,
P. I. (2002) Imaging sites of receptor dephosphorylation by PTP1B on
the surface of the endoplasmic reticulum. Science 295, 1708−1711.
(65) Lee, S. R., Kwon, K. S., Kim, S. R., and Rhee, S. G. (1998)
Reversible inactivation of protein-tyrosine phosphatase 1B in A431
cells stimulated with epidermal growth factor. J. Biol. Chem. 273,
15366−15372.
(66) Leslie, N. R., Bennett, D., Lindsay, Y. E., Stewart, H., Gray, A.,
and Downes, C. P. (2003) Redox regulation of PI 3-kinase signalling
via inactivation of PTEN. EMBO J. 22, 5501−5510.
(67) Maehama, T., Taylor, G. S., and Dixon, J. E. (2001) PTEN and
myotubularin: Novel phosphoinositide phosphatases. Annu. Rev.
Biochem. 70, 247−279.
(68) Lee, S. R., Yang, K. S., Kwon, J., Lee, C., Jeong, W., and Rhee, S.
G. (2002) Reversible inactivation of the tumor suppressor PTEN by
H2O2. J. Biol. Chem. 277, 20336−20342.
(69) Kwon, J., Lee, S. R., Yang, K. S., Ahn, Y., Kim, Y. J., Stadtman, E.
R., and Rhee, S. G. (2004) Reversible oxidation and inactivation of the
tumor suppressor PTEN in cells stimulated with peptide growth
factors. Proc. Natl. Acad. Sci. U.S.A. 101, 16419−16424.
(70) Agazie, Y. M., and Hayman, M. J. (2003) Molecular mechanism
for a role of SHP2 in epidermal growth factor receptor signaling. Mol.
Cell. Biol. 23, 7875−7886.
(71) Paulsen, C. E., Truong, T. H., Garcia, F. J., Homann, A., Gupta,
V., Leonard, S. E., and Carroll, K. S. (2012) Peroxide-dependent
9964
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965
Biochemistry
Current Topic
(91) Wood, Z. A., Schroder, E., Robin Harris, J., and Poole, L. B.
(2003) Structure, mechanism and regulation of peroxiredoxins. Trends
Biochem. Sci. 28, 32−40.
(92) Kang, S. W., Chae, H. Z., Seo, M. S., Kim, K., Baines, I. C., and
Rhee, S. G. (1998) Mammalian peroxiredoxin isoforms can reduce
hydrogen peroxide generated in response to growth factors and tumor
necrosis factor-α. J. Biol. Chem. 273, 6297−6302.
(93) Handy, D. E., Lubos, E., Yang, Y., Galbraith, J. D., Kelly, N.,
Zhang, Y. Y., Leopold, J. A., and Loscalzo, J. (2009) Glutathione
peroxidase-1 regulates mitochondrial function to modulate redoxdependent cellular responses. J. Biol. Chem. 284, 11913−11921.
(94) Halvey, P. J., Watson, W. H., Hansen, J. M., Go, Y. M., Samali,
A., and Jones, D. P. (2005) Compartmental oxidation of thioldisulphide redox couples during epidermal growth factor signalling.
Biochem. J. 386, 215−219.
(95) Winterbourn, C. C. (2008) Reconciling the chemistry and
biology of reactive oxygen species. Nat. Chem. Biol. 4, 278−286.
(96) Parsonage, D., Karplus, P. A., and Poole, L. B. (2008) Substrate
specificity and redox potential of AhpC, a bacterial peroxiredoxin. Proc.
Natl. Acad. Sci. U.S.A. 105, 8209−8214.
(97) Peskin, A. V., Low, F. M., Paton, L. N., Maghzal, G. J., Hampton,
M. B., and Winterbourn, C. C. (2007) The high reactivity of
peroxiredoxin 2 with H2O2 is not reflected in its reaction with other
oxidants and thiol reagents. J. Biol. Chem. 282, 11885−11892.
(98) Woo, H. A., Yim, S. H., Shin, D. H., Kang, D., Yu, D. Y., and
Rhee, S. G. (2010) Inactivation of peroxiredoxin I by phosphorylation
allows localized H2O2 accumulation for cell signaling. Cell 140, 517−
528.
(99) Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) Affinity
labeling of the protein kinase associated with the epidermal growth
factor receptor in membrane vesicles from A431 cells. J. Biol. Chem.
257, 4019−4022.
(100) Clark, S., and Konstantopoulos, N. (1993) Sulphydryl agents
modulate insulin- and epidermal growth factor (EGF)-receptor kinase
via reaction with intracellular receptor domains: Differential effects on
basal versus activated receptors. Biochem. J. 292 (Part 1), 217−223.
(101) Woltjer, R. L., and Staros, J. V. (1997) Effects of sulfhydryl
modification reagents on the kinase activity of the epidermal growth
factor receptor. Biochemistry 36, 9911−9916.
(102) Seo, Y. H., and Carroll, K. S. (2009) Profiling protein thiol
oxidation in tumor cells using sulfenic acid-specific antibodies. Proc.
Natl. Acad. Sci. U.S.A. 106, 16163−16168.
(103) Reddie, K. G., Seo, Y. H., Muse, W. B., III, Leonard, S. E., and
Carroll, K. S. (2008) A chemical approach for detecting sulfenic acidmodified proteins in living cells. Mol. BioSyst. 4, 521−531.
(104) Leonard, S. E., Reddie, K. G., and Carroll, K. S. (2009) Mining
the thiol proteome for sulfenic acid modifications reveals new targets
for oxidation in cells. ACS Chem. Biol. 4, 783−799.
(105) Seo, Y. H., and Carroll, K. S. (2011) Quantification of Protein
Sulfenic Acid Modifications Using Isotope-Coded Dimedone and
Iododimedone. Angew. Chem., Int. Ed. 50, 1342−1345.
(106) Truong, T. H., Garcia, F. J., Seo, Y. H., and Carroll, K. S.
(2011) Isotope-coded chemical reporter and acid-cleavable affinity
reagents for monitoring protein sulfenic acids. Bioorg. Med. Chem. Lett.
21, 5015−5020.
(107) Leonard, S. E., and Carroll, K. S. (2011) Chemical 'omics'
approaches for understanding protein cysteine oxidation in biology.
Curr. Opin. Chem. Biol. 15, 88−102.
(108) Benitez, L. V., and Allison, W. S. (1974) The inactivation of
the acyl phosphatase activity catalyzed by the sulfenic acid form of
glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins.
J. Biol. Chem. 249, 6234−6243.
(109) Saxon, E., and Bertozzi, C. R. (2000) Cell surface engineering
by a modified Staudinger reaction. Science 287, 2007−2010.
(110) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K.
B. (2002) A stepwise Huisgen cycloaddition process: Copper(I)catalyzed regioselective “ligation” of azides and terminal alkynes.
Angew. Chem., Int. Ed. 41, 2596−2599.
(111) Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer
with small molecule kinase inhibitors. Nat. Rev. Cancer 9, 28−39.
(112) Singh, J., Petter, R. C., and Kluge, A. F. (2010) Targeted
covalent drugs of the kinase family. Curr. Opin. Chem. Biol. 14, 475−
480.
(113) Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The
resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307−317.
(114) Yun, C. H., Mengwasser, K. E., Toms, A. V., Woo, M. S.,
Greulich, H., Wong, K. K., Meyerson, M., and Eck, M. J. (2008) The
T790M mutation in EGFR kinase causes drug resistance by increasing
the affinity for ATP. Proc. Natl. Acad. Sci. U.S.A. 105, 2070−2075.
(115) Yun, C. H., Boggon, T. J., Li, Y., Woo, M. S., Greulich, H.,
Meyerson, M., and Eck, M. J. (2007) Structures of lung cancer-derived
EGFR mutants and inhibitor complexes: Mechanism of activation and
insights into differential inhibitor sensitivity. Cancer Cell 11, 217−227.
(116) Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J.
(2006) An allosteric mechanism for activation of the kinase domain of
epidermal growth factor receptor. Cell 125, 1137−1149.
(117) Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst,
P., and Snyder, S. H. (2001) Protein S-nitrosylation: A physiological
signal for neuronal nitric oxide. Nat. Cell Biol. 3, 193−197.
(118) Jaffrey, S. R., and Snyder, S. H. (2001) The biotin switch
method for the detection of S-nitrosylated proteins. Sci. STKE 2001,
pl1.
(119) Wang, H., and Xian, M. (2011) Chemical methods to detect Snitrosation. Curr. Opin. Chem. Biol. 15, 32−37.
(120) Giustarini, D., Dalle-Donne, I., Colombo, R., Milzani, A., and
Rossi, R. (2008) Is ascorbate able to reduce disulfide bridges? A
cautionary note. Nitric Oxide 19, 252−258.
(121) Estrada, C., Gomez, C., Martin-Nieto, J., De Frutos, T.,
Jimenez, A., and Villalobo, A. (1997) Nitric oxide reversibly inhibits
the epidermal growth factor receptor tyrosine kinase. Biochem. J. 326
(Part 2), 369−376.
(122) Murillo-Carretero, M., Torroglosa, A., Castro, C., Villalobo, A.,
and Estrada, C. (2009) S-Nitrosylation of the epidermal growth factor
receptor: A regulatory mechanism of receptor tyrosine kinase activity.
Free Radical Biol. Med. 46, 471−479.
(123) Lam, Y. W., Yuan, Y., Isaac, J., Babu, C. V., Meller, J., and Ho,
S. M. (2010) Comprehensive identification and modified-site mapping
of S-nitrosylated targets in prostate epithelial cells. PLoS One 5, e9075.
(124) Glynn, S. A., Boersma, B. J., Dorsey, T. H., Yi, M., Yfantis, H.
G., Ridnour, L. A., Martin, D. N., Switzer, C. H., Hudson, R. S., Wink,
D. A., Lee, D. H., Stephens, R. M., and Ambs, S. (2010) Increased
NOS2 predicts poor survival in estrogen receptor-negative breast
cancer patients. J. Clin. Invest. 120, 3843−3854.
(125) Switzer, C. H., Glynn, S. A., Cheng, R. Y., Ridnour, L. A.,
Green, J. E., Ambs, S., and Wink, D. A. (2012) S-Nitrosylation of
EGFR and Src Activates an Oncogenic Signaling Network in Human
Basal-Like Breast Cancer. Mol. Cancer Res. 10, 1203−1215.
(126) Penel, S., Hughes, E., and Doig, A. J. (1999) Side-chain
structures in the first turn of the α-helix. J. Mol. Biol. 287, 127−143.
(127) Anderson, T. A., and Sauer, R. T. (2003) Role of an Ncap
residue in determining the stability and operator-binding affinity of Arc
repressor. Biophys. Chem. 100, 341−350.
(128) Miranda, J. J. (2003) Position-dependent interactions between
cysteine residues and the helix dipole. Protein Sci. 12, 73−81.
(129) Kemble, D. J., and Sun, G. (2009) Direct and specific
inactivation of protein tyrosine kinases in the Src and FGFR families
by reversible cysteine oxidation. Proc. Natl. Acad. Sci. U.S.A. 106,
5070−5075.
(130) Singh, J., Dobrusin, E. M., Fry, D. W., Haske, T., Whitty, A.,
and McNamara, D. J. (1997) Structure-based design of a potent,
selective, and irreversible inhibitor of the catalytic domain of the erbB
receptor subfamily of protein tyrosine kinases. J. Med. Chem. 40,
1130−1135.
(131) Leonard, S. E., Garcia, F. J., Goodsell, D. S., and Carroll, K. S.
(2011) Redox-based probes for protein tyrosine phosphatases. Angew.
Chem., Int. Ed. 50, 4423−4427.
9965
dx.doi.org/10.1021/bi301441e | Biochemistry 2012, 51, 9954−9965